Preparation of specimen arrays on an em grid

ABSTRACT

The invention provides systems or apparatuses for dispensing aqueous materials for electron microscopy (EM). The systems allow dispensing of aqueous materials onto an EM sample grid at individual specimen locations in an ordered array of specimen locations, with each individual specimen location in the array of locations. The systems contain a holder for reversibly receiving an EM sample grid, and a dispenser containing one or more dispensing elements that are configured to discretely dispense one or more aqueous solutions from the dispensing elements onto a plurality of individual specimen locations. The dispenser is able to provide an ordered array of discrete specimen locations discontinuous with one another. In the systems, at least one dispensing element is configured to dispense picoliter volumes of one or more of the aqueous solutions. Additionally, the systems contain a drive mechanism to position the EM sample grid relative to the one or more dispensing elements, as well as one or more reservoirs operably linked to the dispenser for holding the one or more aqueous solutions to be discretely dispensed onto each individual specimen location in the array of locations.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject patent application is a divisional of U.S. patentapplication Ser. No. 14/372,274, filed Jul. 15, 2014 (now pending),which is a national stage application of International Application No.PCT/US2013/000018, filed Jan. 14, 2013 (now abandoned), which claims thebenefit of priority to U.S. Provisional Patent Application No.61/632,046, filed Jan. 17, 2012 (now expired). The full disclosures ofthe priority applications are incorporated herein by reference in theirentirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under RR017573 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Screening of samples using transmission electron microscopy (EM) isoften used in to characterize proteins such as antibodies used fortherapeutics, viruses or virus like particles used in vaccines, drugdelivery particles, or other formulations of nanoparticles. Screeningmay also be used to determine optimal conditions for 2D and 3Dcrystallization of proteins, optimizing preparation conditions for anovel protein or other macromolecular complexes, as well as formulationoptimization.

EM applications such as those discussed above often require the analysisof a large number of conditions in parallel. EM sample preparationtypically requires a cumbersome procedure of obtaining several negativestained samples on EM grid supports for each condition. EM gridstypically comprise of a 3 mm diameter copper mesh (˜25 μm thick) withopen square windows (30 to 200 μm wide) acting as the base substrate. Athin (typically 5-50 nm) carbon film is layered on top this substrate,creating electron transparent (carbon film) regions in the open windows.Other EM grid substrate materials are usually made of other metals orsemiconductor materials such as silicon, with films made of SiliconNitride, Silicon Dioxide or Silicon Carbide. By way of example, eachcondition being analyzed can require (i) plasma treating one or moregrids to create a hydrophilic specimen surface, (ii) pipetting 2-3 μL ofthe appropriate sample in an appropriate buffer onto the specimensurface, (iii) blotting the grid using filter paper to remove excesssample, (iv) pipetting 2-3 μL of stain immediately onto the specimensurface to avoid sample drying, (v) blotting the grid again using filterpaper to remove excess stain, and (vi) allowing the stain to dry. Inmany cases staining using this process requires optimization of severalconditions, such as concentration (sample and stain), bufferconstituents, pH, sample and stain application time. This results in alarge number of grid trials, which entail loading individual grids intothe electron microscope for analysis of each condition, and wastes largevolumes of what are often very precious sample material.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositionspreparation of complex specimen arrays for analysis by electronmicroscopy. These methods and compositions can permit high throughputscreening of samples on single EM grid supports using sample volumes inthe nanoliter and picoliter range (required for rare and difficult toobtain samples). Because picoliter scale volumes will not cover anentire EM sample grid surface under achievable contact angles, each EMsample grid may be processed to provide an array of specimen locationson a single grid.

In a first aspect, the present invention provides methods for preparingan electron microscopy sample on an EM sample grid. The methodscomprise:

a. dispensing a plurality of discrete specimens onto an EM sample gridin an ordered array of specimen locations, each specimen of theplurality of discrete specimens being placed into an individual specimenlocation in the array of locations, and each individual specimenlocation in the array of locations having an area of between about 2000μm² to about 70,000 μm² (for example, each location is a circular spotof between about 50 μm and about 300 μm in diameter); and

b. applying a discrete volume of a stain material suitable for contrastenhancement in an electron microscope to each individual specimenlocation in the array of locations that received a specimen.

In certain embodiments, a wash step precedes the application of stain tothe specimen locations. In preferred embodiments, excess wash materialis removed with a porous or bibulous material, or microstructures thatinduce local capillary effects, that “blots” or “wicks” material awayfrom the specimen location. By way of example such a method maycomprise:

a. dispensing a plurality of discrete specimens onto an EM sample gridin an ordered array of specimen locations, each specimen of theplurality of discrete specimens being placed into an individual specimenlocation in the array of locations, and each individual specimenlocation in the array of locations having an area of between about 2000μm² to about 70,000 μm² (for example, each location is a circular spotof between about 50 μm and about 300 μm in diameter);

b. applying a discrete volume of a wash solution to each individualspecimen location in the array of locations that received a specimen,and removing excess wash solution by contact with a bibulous or porousmaterial at the periphery of each individual specimen location in thearray of locations; and

c. applying a discrete volume of a stain material suitable for contrastenhancement in an electron microscope to each individual specimenlocation in the array of locations that received a specimen.

Suitable stains for use in the present methods include ammoniummolybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmiumtetroxide, osmium ferricyanide and auroglucothionate, commerciallyavailable stains such as NANOVAN™ Methylamine Vanadate (Nanoprobes,Inc.) and NANO-W™ Methylamine Tungstate (Nanoprobes, Inc.) as well asother stains that are described in the general literature.

Creation of the blotting regions on the grids can be performed by avariety of fabrication techniques. The material used as the blottingmaterial can be patterned by microfabrication techniques on the grid. Byway of example only, thin film blotting material (such as dried gels,adsorption papers and porous membranes) can be laser machined and thenadhered to the surface of a grid. In another example, the blottingmaterial in liquid form can be printed using inkjet printing or stampedusing soft-contact lithography, and then desiccated. Other methods caninclude creating nano-wires and polymer-matrixes by first forming apatterned seed layer and subsequent deposition/polymerization. This listis not meant to be limiting.

In various embodiments, the sample volume(s) are applied to subregionsof the EM sample grid surface by spotting such as by the use of roboticmicropipetting techniques, or more preferably using “ink jet” printingtechnologies. Ink jet printing technologies known in the art includedevices equipped with pins, or sample ejection elements that dispenseusing thermal, sonic, or piezoelectric impulses. Among the methodsmentioned above, the inkjet method is a preferred sample applicationmethod because of its ability to carry out high-density, precisespotting. As used herein, the term “picoliter volumes” refers to avolume of liquid that is at least 1 pL and which is less than 1 nL;“nanoliter volumes” refers to a volume of liquid that is at least 1 nLand which is less than 1 μL; and “microliter volumes” refers to a volumeof liquid that is at least 1 μL and which is less than 1 mL.

The inkjet method is a method in which a solution of interest is placedin an extra-fine nozzle, pressure or heat is instantaneously applied ona portion near the nozzle's tip to correctly eject an extremely lowvolume of aqueous material from the nozzle's tip and directed to thesurface of the EM grid. For example, the inkjet head may be a bubble-jethead having a mechanism for discharging a solvent with the applicationof thermal energy; a piezo-jet head that ejects a solution using apiezoelectric element; etc. Preferably, the drop dispenser comprisesmultiple nozzles that can be used to “print” sample volumes, washsolutions, stains, etc., onto the discretely addressable samplelocations of the EM sample grid. When the aqueous solution is ejectedfrom the inkjet head, each droplet forms a circular spot, the thicknessand expansion of which is controlled by the structure of the EM gridsurface. Connection with an adjacent spot can be effectively preventedeven when the spots of sample solution are spotted in high density. On astandard ˜3 mm diameter EM grid (2 mm diameter imaging area), when thedispensed spots are 50-300 μm in diameter, transfer of the entirecontents of the 12, 24, 48, 96 or 384 well-plate is possible.

In a related aspect, the present invention relates to EM specimen gridsthat are configured for use in the present invention. The EM specimengrids comprise a plurality of discrete specimen locations delimited fromone another by peripheral regions comprising a bibulous or porousmaterial, or microstructures that induce local capillary effects, that“blots” or “wicks” material. These grids are referred to herein as“blotting microwell array” or “BMA” grids.

In another related aspect, the present invention relates to a system fordispensing aqueous materials onto an EM sample grid at individualspecimen locations in an ordered array of specimen locations, eachindividual specimen location in the array of locations having an area ofbetween about 2000 μm² to about 70,000 μm². The systems comprise:

a. a holder for reversibly receiving an EM sample grid;

b. a picoliter to nanoliter volume drop dispenser (e.g., an inkjetprinting element) configured to dispense fluid from one or moredispensing elements onto each individual specimen location in the arrayof locations;

c. a drive mechanism to position the EM sample grid relative to the oneor more dispensing elements; and

d. one or more reservoirs operably linked to the drop dispenser forholding one or more aqueous solutions to be dispensed onto eachindividual specimen location in the array of locations.

The present invention is particularly applicable to transmissionelectron microscopy. Transmission electron microscopy (TEM) is amicroscopy technique whereby a beam of electrons is transmitted throughan ultra thin specimen, interacting with the specimen as it passesthrough. An image is formed from the interaction of the electronstransmitted through the specimen; the image is magnified and focusedonto an imaging device, such as a fluorescent screen, on a layer ofphotographic film, or to be detected by a sensor such as a CCD camera.The image is in effect assumed to be a simple two-dimensional projectionof the sample down the optical axis.

The methods and compositions of the present invention may be used toanalyze particles selected from the group consisting of polymer beads,metal beads, proteins, protein-metal bead complexes, protein-polymerbead complexes, viruses, virus-like particles, liposomes and othernanoparticles. The methods may also be used to analyze sub micronaggregates of these particles.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts in schematic form an exemplary method for conducting ahigh-throughput screen of samples on an EM compatible grid according tothe invention.

FIG. 2 depicts in schematic form an exemplary method for dispensing ofsamples and stain on an EM compatible grid according to the invention.

FIG. 3 depicts in schematic form an exemplary method for dispensing ofsamples and stain on an EM compatible grid that comprises an array ofblotting material surrounding the targeted area on the grid according tothe invention.

DETAILED DESCRIPTION OF THE INVENTION

Molecular microscopy is a non-invasive molecular imaging technology thatuses advanced specimen preparation and imaging methods designedspecifically to visualize complex biological samples, under conditionsclose to their native state. For well-ordered samples such as viruses,and virus-antibody complexes, the achievable resolution can be <0.4 nm.High-throughput molecular microscopy combines robotic instruments,automated data collection and processing software, and a relationaldatabase into a pipeline to prepare, image, and analyze samples in areproducible manner and with throughputs capable of addressingbiopharmaceutical characterization needs in a statistically significantmanner. Samples are preserved in solution by vitrification (using anautomated cryogenic robot) or by negative stain, and then imaged using atransmission electron microscope (TEM) controlled by automated softwarethat enables sampling of a significant portion of the specimen. Data isanalyzed and stored in a secure database that tracks all aspects ofsample preparation, imaging, and analysis to provide our currentcustomers with a tightly controlled system for biological imaging.

In electron microscopy, staining is usually done with heavy metal saltscommonly derived from molybdenum, uranium, or tungsten. Heavy ions areused since they will readily interact with the electron beam and produceamplitude contrast. A small drop of the sample is deposited on thecarbon coated grid, allowed to settle for approximately one minute,blotted dry if necessary, and then covered with a small drop of thestain (for example 2% uranyl acetate). After a few seconds, this drop isalso blotted dry, and the sample is ready to be imaged in the TEM.

The present invention here describes methods and compositions forconducting a high-throughput screen of samples on a single EM compatiblegrid. As shown in FIG. 1, a standard well-plate (96 or 384 wells)contains the sample conditions to be tested (in lower throughput screens12, 24 and 48 well-plates can also be accommodated). An inkjet headcapable of delivering samples (picoliters to microliters) transfers thesample conditions from the stock plate onto a targeted area of a singleEM grid. The dispensed samples are registered precisely for downstreamidentification and tracking during EM imaging at low and highmagnification. Multiple inkjet heads can be used to facilitate sampledispensing onto the EM grid. On a 3 mm diameter grid (2 mm imagingarea), when the dispensed spots are 50-300 μm in diameter, transfer ofthe entire contents of the 12, 24, 48, 96 or 384 well-plate is possible.This allows for complete mapping of sample conditions from the standardwell-plate onto the grid. For screens requiring more thousands of sampleconditions, only a few EM grids will be required.

In one scenario of the invention, the samples are dispensed and dried onthe grid prior to any staining. This scenario can be used if the samplesare relatively stable and the drying (accompanied by phenomenon such assalt crystallization) does not lead to particle destabilization orstaining failure. In such situations, once inkjet sample transfer iscomplete, the grid can be washed and flooded with stain (3 μL).Alternatively, the stain can be dispensed onto the individual samplespots on the grid using a single inkjet head that precisely targets theregistered areas. As shown in FIG. 2, if multiple heads are used, thedispensing of stain can take place before the dispensed sample dries. Ineither case, dispensing of stain using an inkjet head allows for muchgreater control of volume and uniformity of spreading across the grid,which is not possible with the standard blotting process. Additionally,multiple staining conditions (concentration and type of stain) can betested on similar sample conditions. Multiplexing at the grid levelallows only a single grid (or a few, compared to hundreds to thousands)to be loaded in the electron microscope for the screen.

Sample constituents can include dissolvable materials such as sugars,gels and buffer salts that prevent the destabilization of sensitivesamples during the brief period of evaporation after the first dropletlands and spreads on the grid. As shown in FIG. 2, diffusion of thestain particles occurs after the second droplet lands on the samplespot. Along with the spatial precision of droplet transfer, the timeinterval between the first and second droplet can also be accuratelycontrolled within a few hundred milliseconds to seconds. Multipledispense heads can allow for intermediate washes, bindings andreactions, between the sample and stain droplet. The surface propertiesof the grid (flatness, wetability and atomic roughness) govern thespreading of droplets given comparable environmental conditions. Thegrid surface can be made hydrophilic (or super-hydrophilic) to ensurerapid spreading of the droplets and faster diffusion between the sampleand stain.

To further control sample washing and staining without significantevaporation prior to drying, an array of blotting material can surroundthe targeted area on the grid as shown in FIG. 3. After sample spotting,the wash and stain steps with larger dispense volume leads to localblotting in the surrounding material. In this manner the samples can bewashed, without significant buildup in the target area. Similarly, thesubsequent dispensed stain will be blotted locally to create an evenlayer of negatively stained sample. As noted above, the material used asthe blotting material can be patterned by microfabrication techniques onthe grid. In one method, thin film blotting material (such as driedgels, adsorption papers or porous membranes) can be laser machined andthen adhered to the surface of a grid. In another method, the blottingmaterial in liquid form can be printed using inkjet printing or stampedusing soft-contact lithography, and then desiccated. Other methods caninclude creating nano-wires and polymer-matrixes by first forming apatterned seed layer and subsequent deposition/polymerization. Othermethods can include creating microstructures, surrounding the targetedareas that induce local capillary effects, such as an overhanging ledgeor spiral with spaces of 0.5 to 10 μm between hydrophilic walls. The BMAgrids can be aligned accurately with the inkjet printer to dispense thedroplets between the blotting areas. The blotting areas themselves canbe used as physical markers for identifying the registered samples andfor downstream image recognition and processing.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationsthat is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions that have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A system for dispensing aqueous materials onto anEM sample grid at individual specimen locations in an ordered array ofspecimen locations, each individual specimen location in the array oflocations, comprising: a holder for reversibly receiving an EM samplegrid; a dispenser comprising one or more dispensing elements andconfigured to discretely dispense one or more aqueous solutions from theone or more dispensing elements onto a plurality of individual specimenlocation to thereby provide an ordered array of discrete specimenlocations discontinuous with one another, wherein at least onedispensing element is configured to dispense picoliter volumes of one ormore of the aqueous solutions; a drive mechanism to position the EMsample grid relative to the one or more dispensing elements; and one ormore reservoirs operably linked to the dispenser for holding the one ormore aqueous solutions to be discretely dispensed onto each individualspecimen location in the array of locations.
 2. A system according toclaim 1, wherein the one or more of the dispensing elements comprises athermal dispensing element.
 3. A system according to claim 1, whereinthe one or more of the dispensing elements comprises a piezoelectricdispensing element.
 4. A system according to claim 1, wherein the one ormore of the dispensing elements comprises a sonic dispensing element. 5.A system according to claim 1, wherein the system is configured todiscretely dispense a first aqueous solution comprising a test samplefrom one or more of the dispensing elements onto the plurality ofindividual specimen locations, and to subsequently discretely dispense asecond aqueous solution comprising a stain material suitable forcontrast enhancement in an electron microscope from one or more of thedispensing elements onto the plurality of individual specimen locations.6. A system according to claim 2, wherein the system is configured todiscretely dispense a first aqueous solution comprising a test samplefrom one or more of the dispensing elements onto the plurality ofindividual specimen locations, and to subsequently discretely dispense asecond aqueous solution comprising a stain material suitable forcontrast enhancement in an electron microscope from one or more of thedispensing elements onto the plurality of individual specimen locations.7. A system according to claim 3, wherein the system is configured todiscretely dispense a first aqueous solution comprising a test samplefrom one or more of the dispensing elements onto the plurality ofindividual specimen locations, and to subsequently discretely dispense asecond aqueous solution comprising a stain material suitable forcontrast enhancement in an electron microscope from one or more of thedispensing elements onto the plurality of individual specimen locations.8. A system according to claim 4, wherein the system is configured todiscretely dispense a first aqueous solution comprising a test samplefrom one or more of the dispensing elements onto the plurality ofindividual specimen locations, and to subsequently discretely dispense asecond aqueous solution comprising a stain material suitable forcontrast enhancement in an electron microscope from one or more of thedispensing elements onto the plurality of individual specimen locations.